Nanog Protein Research Articles

Generation of Melanocytes from Induced Pluripotent Stem Cells

Epidermal melanocytes play an important role in protecting skin from ultraviolet (UV) rays, and are implicated in a variety of skin diseases. Here, we developed an efficient method for differentiating induced pluripotent stem cells (iPSCs) into melanocytes. We first generated iPSCs from adult mouse tail-tip fibroblasts (TTFs) using retroviral vectors or virus-free piggyBac transposon vectors carrying murine Sox2, Oct3/4, cMyc and Klf4. The TTF-derived iPSC clones exhibited similar morphology and growth properties as mouse embryonic stem (ES) cells. The iPSCs expressed ES cell markers, displayed characteristic epigenetic changes and formed teratomas with all three germ layers. The iPSCs were used to generate embryoid bodies (EBs) and were then successfully differentiated into melanocytes by treatment with growth factors. The iPSC-derived melanocytes expressed characteristic melanocyte markers and produced melanin pigment. Electron microscopy showed that the melanocytes contained mature melanosomes. We manipulated the conditions used to differentiate iPSCs to melanocytes and discovered that Wnt3a is not required for mouse melanocyte differentiation. This report shows that melanocytes can be readily generated from iPSCs, providing a powerful resource for the in vitro study of melanocyte developmental biology and diseases. By inducing iPSCs without viruses, the possibility of integration mutagenesis is alleviated, providing iPSCs are more compatible for cell replacement therapies.

Abstract C115: Anticancer mechanism of Rg3-NPs nanoparticles in primary cancer cells and embryonal carcinoma.

Abstract Ginsenoside Rg3[20(R)-Ginsenoside Rg3] is a natural active ingredient that is extracted from Korean red ginseng root. Ginsenoside Rg3 elevates therapeutic effect of radiotherapy and chemotherapy. The use of Rg3 as a potential anti-cancer durg has been addressed in some papers, but Pang et al found that the application of ginsenoside Rg3 is heavily limited by its low bioavailability and poor absorption via oral administration. To overcome these problems, a new microencapsulated drug delivery system is needed that can control drug delivery and extend the drug circulation time in blood. In the present study, Rg3-loaded PEG-PLGA-NPs (Rg3-NPs) were prepared by the modified spontaneous emulsification solvent diffusion (SESD) method, and the physicochemical characteristics of Rg3-NPs were investigated. We treated primary glioblastoma, hepatocellular carcinoma and human embryonic carcinoma cells (NCCIT) with 50μM Rg3-NPs for 48h. We then used gene expression arrays (Illumina) for genome-wide expression analysis and validated the results for genes of interest by means of Real-Time PCR. Functional annotations were then performed using the DAVID and KEGG online tools. We compared the expression profiles of NCCIT cells with that of the cancer cell lines in order to identify common Rg3-NPs -dependent transcriptional changes and accompanying signaling cascades. The results show that the Rg3-NPs are slick and uniform, the average diameter of nanopartical is between 75–90nm, and entrapment efficiency is (89.7×1.7)%. MTT shows that the growth of cells can be significantly inhibited by Rg3-NPs in a dose-dependence manner. FCM test shows Rg3-NPs can be released from the conjugate nanopartical and react with the genes in the cell nuclei causing changes in the gene molecules. We also found that cancer cells treated with Rg3-NPs undergo cell-cycle arrest at different checkpoints. This arrest was associated with a decrease in the mRNA levels of core regulatory genes, BUB1, CDC20, TTKand CENPE, as determined by microarray-analysis and verified by RealTime PCR. In contrast, human NCCIT cells showed over-expression of GADD45 A and G (growth arrest- and DNA-damage-inducible proteins 45A and G), as well as down-regulation of OCT4, and NANOG protein. Furthermore, Rg3-NPs induced the expression of apoptotic and anti-migratory proteins p53 in all cell lines. Rg3-NPs also up-regulated steady-state levels of both cyclin A and B. The results of the present study, together with the results of earlier studies show that Rg3-NPs targets genes involved in the progression of the M-phase of the cell cycle. It is associated with several important pathway, which include apoptosis (p53) and motility (p38; MAPK). Rg3-NPs may be a potent cell-cycle regulation drug targeting the M-phase, both in cell lines and primary patient-derived cancer cells from various tumor entities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C115.

Wnt/β‐catenin signaling is critical for dedifferentiation of aged epidermal cells in vivo and in vitro

Aged epidermal cells have the capacity to dedifferentiate into stem cell-like cells. However, the signals that regulate the dedifferentiation of aged epidermal cells remain unclear. Here, we provide evidence that Wnt/β-catenin is critical for aged epidermal cell dedifferentiation in vivo and in vitro. Some aged epidermal cells in human ultrathin epidermal sheets lacking basal stem cells transplanted onto wounds dedifferentiated into stem cell-like cells that were positive for CK19 and β1 integrin but negative for CK10. In addition, Wnt/β-catenin pathway was activated during this process. There was increased expression of Wnt-1, Wnt-4, Wnt-7a, β-catenin, cyclin D1, and c-myc. Secreted frizzled-related protein 1, a Wnt/β-catenin pathway inhibitor, blocked dedifferentiation in vivo. Then, the activator, a highly specific glycogen synthase kinase (GSK)-3β inhibitor, of Wnt/β-catenin pathway was added to the culture medium of aged epidermal cells. Surprisingly, we found that the activator induced higher expression of CK19, β1 integrin, Oct4, and Nanog proteins. The induced aged epidermal cells exhibited high colony-forming efficiency, long-term proliferative potential and could regenerate a skin equivalent (as do epidermal stem cells). These results suggested that activation of Wnt/β-catenin pathway induced the dedifferentiation of aged epidermal cells, which suggest a new approach to generate epidermal stem cell-like cells.

The seminoma cell line TCam‐2 is sensitive to HDAC inhibitor depsipeptide but tolerates various other chemotherapeutic drugs and loss of NANOG expression

Seminomas and embryonal carcinomas (EC) are both type II germ cell tumor (GCT) entities and develop from the same precursor lesion (carcinoma-in situ, CIS). However, they show significant differences in growth behavior, differentiation potential, and gene expression. Although ECs are prone to differentiate into all three germ layers and give rise to the non-seminomatous GCT entities teratoma, choriocarcinoma, and yolk-sac tumor, differentiation of seminomas to these entities is only rarely observed. This might reflect the ability of seminomas to actively inhibit differentiation processes evoked by environmental cues. Also, it is not known why CIS gives rise to seminoma in some patients and to non-seminoma in the others. Here, we treated the seminoma-like cell line TCam-2 with the HDAC-inhibitor Depsipeptide, the global demethylating agent 5-aza-2'-deocycytidine, all-trans retinoic acid and the monaminooxidase inhibitor Tranylcipromine and also used knock down approaches to reduce expression of the pluripotency marker NANOG and/or the inhibitor of primordial germ cell differentiation TFAP2C. We found that TCam-2 cells induce apoptosis when treated with Depsipeptide (> 10 nM) but are resistant to treatments with 5-aza-2'-deocycytidine, all-trans retinoic acid and Tranylcipromine, highlighting Depsi as a treatment option for seminomas. We show that TCam-2 cells up-regulate endoderm- and throphectoderm-associated genes after down-regulation of NANOG expression; however, morphologically no indications of differentiation could be found. Instead, we observed up-regulation of OCT3/4 and SOX17 in TCam-2-NANOG knockdown and speculate that this compensates for the loss of the NANOG protein. Hence, NANOG is not a primary target gene responsible for the inhibition of differentiation in seminomas.

Brief Report: Benchmarking Human Pluripotent Stem Cell Markers During Differentiation Into the Three Germ Layers Unveils a Striking Heterogeneity: All Markers Are Not Equal

Pluripotent stem cells (PSC) are functionally characterized by their capacity to differentiate into all the cell types from the three germ layers. A wide range of markers, the expression of which is associated with pluripotency, has been used as surrogate evidence of PSC pluripotency, but their respective relevance is poorly documented. Here, we compared by polychromatic flow cytometry the kinetics of loss of expression of eight widely used pluripotency markers (SSEA3, SSEA4, TRA-1-60, TRA-1-81, CD24, OCT4, NANOG, and alkaline phosphatase [AP]) at days 0, 5, 7, and 9 after induction of PSC differentiation into cells representative of the three germ layers. Strikingly, each marker showed a different and specific kinetics of disappearance that was similar in all the PSC lines used and for all the induced differentiation pathways. OCT4, SSEA3, and TRA-1-60 were repeatedly the first markers to be downregulated, and their expression was completely lost at day 9. By contrast, AP activity, CD24, and NANOG proteins were still detectable at day 9. In addition, we show that differentiation markers are coexpressed with pluripotency markers before the latter begin to disappear. These results suggest that OCT4, SSEA3, and TRA-1-60 might be better to trace in vitro the emergence of pluripotent cells during reprogramming.

Differentiation-Independent Fluctuation of Pluripotency-Related Transcription Factors and Other Epigenetic Markers in Embryonic Stem Cell Colonies

Embryonic stem cells (ESCs) maintain their pluripotency through high expression of pluripotency-related genes. Here, we show that differing levels of Oct4, Nanog, and c-myc proteins among the individual cells of mouse ESC (mESC) colonies and fluctuations in these levels do not disturb mESC pluripotency. Cells with strong expression of Oct4 had low levels of Nanog and c-myc proteins and vice versa. In addition, cells with high levels of Nanog tended to occupy interior regions of mESC colonies. In contrast, peripherally positioned cells within colonies had dense H3K27-trimethylation, especially at the nuclear periphery. We also observed distinct levels of endogenous and exogenous Oct4 in particular cell cycle phases. The highest levels of Oct4 occurred in G2 phase, which correlated with the pKi-67 nuclear pattern. Moreover, the Oct4 protein resided on mitotic chromosomes. We suggest that there must be an endogenous mechanism that prevents the induction of spontaneous differentiation, despite fluctuations in protein levels within an mESC colony. Based on the results presented here, it is likely that cells within a colony support each other in the maintenance of pluripotency.

A Tcf/Lef element within the enhancer region of the human NANOG gene plays a role in promoter activation

NANOG is a homeodomain-containing transcription factor that is essential for the maintenance of pluripotency and self-renewal in embryonic stem cells. However, the molecular mechanisms underlying the regulation of NANOG expression in human cells remain largely unknown. Here, we investigated the role of Tcf/Lef response elements located in the enhancer of the human NANOG gene. We found that forced expression of Lef1 or β-catenin stimulated human NANOG promoter activity, while shRNA-mediated knockdown of β-catenin reduced Lef1-induced NANOG promoter activation. Deletion or mutation of the Tcf/Lef element within the enhancer region of the human NANOG gene completely abrogated Lef1-induced NANOG promoter activity. The results of a chromatin immunoprecipitation assay demonstrated that Lef1 and β-catenin bind to the Tcf/Lef element in the enhancer region of the NANOG gene. Forced expression of GSK-3β inhibited basal, Lef1-induced, and β-catenin-induced NANOG promoter activity, while treatment with the GSK-3β inhibitor SB216763 resulted in the accumulation of β-catenin and NANOG protein. Furthermore, Dvl-1-induced NANOG promoter activity was abrogated by the expression of β-catenin shRNA. Stable overexpression of Dvl-1 caused β-catenin and NANOG to accumulate. These results indicate that the Tcf/Lef response element in the enhancer region of the human NANOG gene is able to stimulate NANOG gene transcription.

Spermatogonial stem cells in the testis of an endangered bovid: Indian black buck ( Antilope cervicapra L.)

Numerous wild bovids are facing threat of extinction owing to the loss of habitat and various other reasons. Spermatogonial stem cells (SSCs) represent the only germline stem cells in adult body that are capable of self-renewal and that can undergo differentiation to produce haploid germ cells. SSCs can, therefore, serve as a useful resource for preservation of germplasm of threatened and endangered mammals. The Indian black buck ( Antilope cervicapra L.) is a small Indian antelope that is listed as endangered by the Indian Wildlife Protection Act, 1972. Immunohistochemical analysis of testes tissues of black buck revealed the presence of spermatogonia that were specifically stained by lectin- Dolichos biflorus agglutinin (DBA). The expression of pluripotent cell-specific markers, NANOG and stage-specific embryonic antigen-1 (SSEA-1), was detected in spermatogonia. Interestingly, the expression of POU5F1 (OCT3/4) was absent from spermatogonia, however, it was detected in differentiating cells such as spermatocytes and round spermatids but not in elongated spermatids. The expression of NANOG protein was also present in spermatocytes but absent in round and elongated spermatids. Using the testis transplantation assay, stem cell potential of black buck spermatogonia was confirmed as indicated by the presence of colonized DBA-stained cells in the basal membrane of seminiferous tubules of xenotransplanted mice testis. The findings from this study suggest the presence of SSCs in the testis of an endangered bovid for the first time and open new possibility to explore the use of SSCs in conservation.

Expression of NANOG in human gliomas and its relationship with undifferentiated glioma cells

Stemness genes, including NANOG, which have been reported to play a significant role in embryonic stem cells (ESCs), are purported to be expressed in specific human tumor types. In the present study, we explored the expression of NANOG in gliomas to demonstrate its key roles in maintaining the undifferentiated state of glioma cells. Brain tumor stem cells (BTSCs) were isolated from the human glioma cell line U87 and cultured in simplified serum-free medium. Significantly higher NANOG mRNA and protein expression levels were demonstrated in U87 parental attached cells and suspended BTSCs as well as in 69 glioma specimens of different pathological grades. The relative levels of NANOG mRNA and protein expression were higher in the BTSCs as compared to those in the U87 parental attached cells and were significantly positively correlated with pathological grade. The coexpression and relationship of NANOG, CD133 and GFAP in situ in the cellular levels was determined through double-label immunohistochemical staining in the gliomas. A positive correlation of NANOG and CD133 expression with pathological grade of the samples was noted, while NANOG and GFAP expression correlated negatively with the pathological grade (P<0.01). Overexpression of NANOG in gliomas and its close relationship with the undifferentiated state of glioma cells invivo and in vitro indicated that NANOG may contribute to the existence of BTSCs and may be related to tumorigenesis of the cerebrum by maintaining the undifferentiated state of glioma cells, which provides a foundation to further explore its role in the biological behavior of gliomas.

Expression of the stem cell marker, Nanog, in human endometrial adenocarcinoma.

The embryonic stem cell self-renewal gene, Nanog, has been shown to be expressed in several tumor types and to regulate tumor development. The aim of this study was to carry out a detailed analysis of Nanog expression in human endometrial adenocarcinoma (EAC). Immunohistochemical analysis and reverse transcription-polymerase chain reaction were used to characterize Nanog, Sox2, and Oct4 expression in tissue arrays containing EAC, benign endometrium samples, and tumorosphere cells. Tumorosphere formation of EAC-derived cells in the stem cell culture medium was also analyzed. Nanog expression was then analyzed in secondary tumors initiated by the injection of tumorospheres or tumorosphere-derived differentiated cells into 15 female nude mice. Apoptosis and cell proliferation were detected in the fluorescence-activated cell sorter and 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide experiments, respectively. The Nanog protein was expressed in a majority of EAC samples (45 of 55, 81.8%), but not in benign endometrium samples (0 of 26, 0.0%). Oct4 and Sox2 were also commonly expressed in EAC samples (42 of 55, 76.4% and 39 of 55, 70.9%, respectively). Subsets of cancer cells from all EAC samples (15 of 15, 100%) exhibited the capacity to form Nanog-positive tumorospheres. The tumorospheres also expressed Nanog, Oct4, and Sox2 mRNA and showed a higher proliferation potential than differentiated cells. All 15 mice that were injected with tumorosphere cell-formed tumors, whereas only 3 of 15 mice injected with differentiated cells derived from tumorospheres developed tumors. All secondary xenograft tumors still expressed Nanog protein and Nanog, Oct4, and Sox2 mRNA, and had higher proliferation and lower apostosis than did differentiated cells. Overexpression of Nanog in EACs suggests that Nanog may represent a potential therapeutic target for EAC. In addition, Nanog may be useful as a biomarker in an immunohistochemical panel to differentiate between EAC and benign endometrial tissues. The expression of Nanog in tumorospheres may be indicative of the presence of a population of endometrial cancer stem cells, and its expression in xenograft tumors suggests that Nanog may also be associated with tumor metastasis.

Abstract 5559: Long-term exposure of breast cell lines to ethanol affects the transcriptional signature for some oncogenic gene families, but has little effect on this phenotype in mammospheres or on the expression of stem cell markers

Abstract Objectives. Chronic alcoholism is a risk factor for breast cancer but the mechanism of its potential oncogenic effects is unknown. Stem cells have been found in the epithelium of normal breast tissue and are postulated to convert into cancer stem cells driving tumor generation and metastasis. Breast stem cells are assumed to be enriched within multiple-cell spheroids named mammospheres. Here we aimed to determine whether ethanol, or its metabolite, acetaldehyde, stimulate the in vitro proliferation and malignant transformation of human breast stem cells within epithelial cell cultures and mammospheres, and whether this process is defined by specific gene expression signatures denoting oncogenesis, stemness and/or alcohol-regulation. Methods. Cultures of monolayer attached cells and mammospheres from MCF-12 (“normal”) and MCF-7 (“malignant”) cell lines were incubated +/- 25 mM ethanol and subjected to DNA microarray analysis, or in triplicate with increasing concentrations of ethanol (0-25 mM) or acetaldehyde (0-12.5 mM) for 6 days, followed by immunocytofluorescence, quantitative western blot for stem cell markers Oct4 and nanog, and by RT-PCR, the formazan assay and growth in soft agar. Results. Low levels of mRNAs for stem cell markers were seen by DNA microarrays in all cultures, but except for Sox4 and Jag1 were not responsive to ethanol. Oct4 and nanog proteins were detected in the nuclei of MCF-7 attached cells, and expressed respectively as 45 (Oct4a) and 38 kDa bands for the active isoforms, whereas in MCF-12 attached cells they were detected as the variant 33 (Oct4b) and 55 kDa proteins. Ethanol reduced Oct4a moderately in MCF-7 attached cells and acetaldehyde increased it, but no changes occurred in mammospheres. Ethanol increased Oct4b and reduced nanog in MCF-12 attached cells, with no change (Oct4) or an increase (nanog) in mammospheres. In both MCF-7 and MCF-12 attached cells, but not in mammospheres, ethanol upregulated by &amp;gt;2 several alcohol-responsive metallothioneins (mainly 1F, 1X, and 2A), and related trans-membrane proteases serine (TMPRSS), as well as centromer-associated protein and PAI 1, while reducing other serpines and caspase 4, all malignancy-associated genes. Conclusions. Although stem cell markers were expressed in MCF-7 and MCF-12 attached cells, they were not increased in mammospheres, raising doubts about the putative stem cell enrichment in these spheroids. The inconsistent modulation of stem cell number by ethanol and acetaldehyde requires confirmation by cell sorting. The identification of several ethanol-responsive, malignancy-associated genes in both MCF-7 and MCF-12 attached cells may support an oncogenic risk for high alcohol levels. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5559. doi:10.1158/1538-7445.AM2011-5559

PEST Motif Sequence Regulating Human NANOG for Proteasomal Degradation

A number of transcriptional factors are required for pluripotency of stem cells. NANOG, a homeobox transcription factor, plays a critical role in regulating embryonic stem cell (ESC) pluripotency. The expression level of NANOG is tightly regulated, and perturbation in its expression level can lead to significant difference in the morphology, expression of cell surface markers, and growth factor dependence of human and mouse ESCs. Here, we demonstrate that the proteolysis of human NANOG is regulated by the ubiquitin-proteasomal pathway. The inhibition of proteasome activity by proteasome inhibitor MG132 showed increase in protein levels of endogenous NANOG in a dose-dependent manner in human ESCs (hESCS). We demonstrated that the inhibition of the proteasome activity and cotransfection with exogenous ubiquitin promotes endogenous ubiquitination of NANOG by coimmunoprecipitation assay. In addition, we showed that both K48- and K63-branched polyubiquitin chains can conjugate with NANOG in vivo. Moreover, NANOG was an unstable protein and exhibited relatively short half-life of about 120 min in hESCs. Pretreatment of hESCs with proteasome inhibitor MG132 inhibits NANOG protein degradation and extends its half-life. Finally, we found that a PEST motif sequence (rich in proline, glutamine, serine, and threonine) from amino acid 47 to 72 located toward the N-terminus of NANOG was shown to target the protein for degradation. Deletion of the PEST motif reduced ubiquitination of NANOG, leading to NANOG stabilization. Collectively, these results indicate that the expression level, stability, and activity of NANOG are modulated by post-translational mechanisms.

Expression of Nanog in brain tumor stem cells

Objective To detect the expression of Nanog in glioma cell line U87 and the relationship with BTSCs.Methods BTSCs were isolated from glioma cell line U87 and cultured in simplified serum-free neural stem cell medium by nanosphere suspension culture method spheres,and purified continuously through the monoclonal formation experiment.The immunofluorescence staining of cells was employed to identify the BTSCs and differentiated cells.The expression of Nanog mRNA was examined by RT- PCR and Nanog protein was detected by immunocytochemistry in U87 and BTSCs respectively.Double immunocytochemistry staining was used to detect the co- expressions of Nanog/CD133.Methods BTSCs were isolated ,cultured and purified successfully from glioma cell line U87.Both Nanog mRNA and protein were expressed in U87 and BTSCs.The expression rate of immunopositive cells was (64.1 ±18.2)% in U87 and (97.2±1.8)% in BTSCs respectively(t =5.719,P =0.000).The Nanog+/CD133+ cells could be found co-expressed in U87 and BTSCs by using double immunocytochemistry s taining.The relative level of Nanog expression was 0.3851 ±0.0771 in U87 and0.6032±0.1223 in BTSCs(t =4.770,P =0.000).Conclusion BTSCs exist in glioma cell line U87 in vitro.The levels of Nanog expression in BTSCs were higher than that in U87.Nanog expression was associated with the genesis and differentiation state of glioma.The overexpression of Nanog and co- expression of Nanog/CD133 showed that it might contribute to the existence of BTSCs,which lay a foundation to the further explore its role in biological behaviour of glioma.Expression of Nanog in U87 indicated the close relationship between glioma and stem cell,and suggested Nanog may play a critical role in the development of glioma. Key words: Glioma; Gene expression; Nanog; Cancer stem cell

Overexpression of Nanog protein is associated with poor prognosis in gastric adenocarcinoma

Nanog, a key transcription factor in self-renewal and pluripotency of embryonic stem cells, has been proved to play a novel role in solid tumor development. Here, we investigated Nanog protein expression in retrospective clinical samples of 105 patients underwent resective surgery for gastric adenocarcinoma. We found that Nanog protein immunostaining in tumor tissues was stronger than that in their corresponding non-dysplastic tissues. However, no statistical difference of Nanog protein expression between tumor tissues and metastatic lymph nodes was found (P = 0.143). Interestingly, overexpression of Nanog protein was correlated with advanced clinical stage of patients with gastric adenocarcinoma (P = 0.006). And Nanog protein expression was correlated with lymph node status (P = 0.004), infiltrating extent (P = 0.001), and differentiation (P = 0.000) of patients with gastric adenocarcinoma. Survival analysis showed that overexpression of Nanog protein in gastric cancer patients predicted a poorer prognosis (P = 0.000). Our data first demonstrated a potential diagnostic and prognostic role of Nanog for gastric adenocarcinoma.

Expression of stem cell markers in side population cells sorted from SMMC-7721 cell line

Objective To study the expression of stem cell markers in side population cells sorted from SMMC-7721 cell line. Methods Fluorescence-activated cell sorting （FACS） was used to sort side population （SP） cells and non-SP （NSP） cells from SMMC-7721 cell line. Real-time polymerase chain reaction （PCR） and flow cytometry （FCM） were used to evaluate the expression of several stem cell markers such as ABCC2, CD133, Oct4, Sox2 and NANOG in SP cells and NSP cells. Results FACS analysis indicated that （9. 2 ±0. 2） % of the SMMC-7721 cells were SP cells. Real-time PCR analysis suggested that ABCC2, CD133, Oct4, Sox2 and NANOG were expressed in the SP cells at higher levels than the NSP cells by about 7. 132, 4. 985, 8. 642, 5. 095 and 5. 164 folds, respectively （P 〈 0. 01 ）. FCM analysis revealed that the expression of ABCC2, CD133, Oct4, Sox2 and NANOG proteins in SP cells was （92. 65±3.92）%, （12.75 ±1.62）%, （17.35 ±2.31）%, （9.57 ±1.71）% and （28.39 ±5.28）% respectively, while in NSP cells that was （0.26±0.06）%, （2.51±0. 17）%, （1.74±0.38）%, （1.52 ±0.41）% and （3.37 ± 1.02） % respectively （P 〈 0. 01 ）. Conclusion The SP cells sorted from SMMC-7721 cell line may enrich tumor stem cells. Purified liver cancer stem ceils may be obtained by screening SP cells using a variety of stem cell markers. Key words: Carcinoma, hepatocellular; Side population cell; Tumor stem cell

Characteristics of Novel Chicken Embryonic Stem Cells Established Using Chicken Leukemia Inhibitory Factor

Chickens are an ideal animal model study tool for developmental biology, and a farm animal with excellent productivity. Researchers have therefore long sought to establish chicken embryonic stem cells (cESCs) to enable the creation of genetically modified chickens. Here, we derived novel cESCs from chicken blastodermal cells (CBCs) cultured with chicken leukemia inhibitory factor (chLIF). These cESCs have the capacity for long-term (100 days or more) successive subculture and express both chicken Nanog (chNanog) and chicken vasa homolog (Cvh) mRNAs and proteins. The cESCs showed a capacity for chimeric formation during a transplant experiment that used a fertilized egg. Transfer of the enhanced green fluorescent protein (EGFP) gene to cESCs enabled green fluorescence to be observed among primordial germ cells (PGCs). These results indicate that novel cESCs should have the capacity to differentiate into germ cells.

Large Scale Phosphoproteome Profiles Comprehensive Features of Mouse Embryonic Stem Cells

Embryonic stem cells are pluripotent and capable of unlimited self-renewal. Elucidation of the underlying molecular mechanism may contribute to the advancement of cell-based regenerative medicine. In the present work, we performed a large scale analysis of the phosphoproteome in mouse embryonic stem (mES) cells. Using multiplex strategies, we detected 4581 proteins and 3970 high confidence distinct phosphosites in 1642 phosphoproteins. Notably, 22 prominent phosphorylated stem cell marker proteins with 39 novel phosphosites were identified for the first time by mass spectrometry, including phosphorylation sites in NANOG (Ser-65) and RE1 silencing transcription factor (Ser-950 and Thr-953). Quantitative profiles of NANOG peptides obtained during the differentiation of mES cells revealed that the abundance of phosphopeptides and non-phosphopeptides decreased with different trends. To our knowledge, this study presents the largest global characterization of phosphorylation in mES cells. Compared with a study of ultimately differentiated tissue cells, a bioinformatics analysis of the phosphorylation data set revealed a consistent phosphorylation motif in human and mouse ES cells. Moreover, investigations into phosphorylation conservation suggested that phosphoproteins were more conserved in the undifferentiated ES cell state than in the ultimately differentiated tissue cell state. However, the opposite conclusion was drawn from this conservation comparison with phosphosites. Overall, this work provides an overview of phosphorylation in mES cells and is a valuable resource for the future understanding of basic biology in mES cells.

Evolution of the mammalian embryonic pluripotency gene regulatory network

Embryonic pluripotency in the mouse is established and maintained by a gene-regulatory network under the control of a core set of transcription factors that include octamer-binding protein 4 (Oct4; official name POU domain, class 5, transcription factor 1, Pou5f1), sex-determining region Y (SRY)-box containing gene 2 (Sox2), and homeobox protein Nanog. Although this network is largely conserved in eutherian mammals, very little information is available regarding its evolutionary conservation in other vertebrates. We have compared the embryonic pluripotency networks in mouse and chick by means of expression analysis in the pregastrulation chicken embryo, genomic comparisons, and functional assays of pluripotency-related regulatory elements in ES cells and blastocysts. We find that multiple components of the network are either novel to mammals or have acquired novel expression domains in early developmental stages of the mouse. We also find that the downstream action of the mouse core pluripotency factors is mediated largely by genomic sequence elements nonconserved with chick. In the case of Sox2 and Fgf4, we find that elements driving expression in embryonic pluripotent cells have evolved by a small number of nucleotide changes that create novel binding sites for core factors. Our results show that the network in charge of embryonic pluripotency is an evolutionary novelty of mammals that is related to the comparatively extended period during which mammalian embryonic cells need to be maintained in an undetermined state before engaging in early differentiation events.

Special issue for stem cells: a multi-perspective look at stem cells

The past few years may have been a golden time in the history of stem cell research. The unique properties stem cells possess, to proliferate indefinitely and to differentiate into multiple kinds of cells, make these cells wonderful platforms for studying organism development and valuable sources for regenerative medicine. Many research teams worldwide have joined this area and contributed at multiple levels. Nowadays, stem cell research has been an integration of multiple disciplines ranging from basic biological studies of genetic and epigenetic regulation of cell state, to translational biomedical studies on drug discovery and clinical treatment, and to biotechnology such as new biomaterials exploration. Taking the most exciting advances as an example, in 2006 Yamanaka and colleagues generated induced pluripotent stem (iPS) cells by ectopic expression of four transcription factors in somatic cells. iPS cells soon became a focus of mechanistic studies of epigenetic determination of cell fate, such as histone modification, DNA methylation and non-protein-coding RNAs. Meanwhile, disease-specific iPS cells are largely used for high-throughput drug discovery. And many teams have already performed iPS cell-based transplantation therapy in rodent models. Aiming at better utilizing iPS cells, new technologies such as single cell imaging and mRNAmediated gene transfection are being explored. Based on these advancements, we believe there will be more fascinating discoveries in stem cell research in the near future. In this special issue, authors of eleven articles share with us their new findings and ideas on many different aspects of stem cell research. Among them, Cao et al. report that recombinant OCT4 and NANOG proteins have different effects on cultured human fibroblast cells, indicating different mechanisms in epigenetic reprogramming. Xiao et al. report their screening for self-renewal factors using a combination of mRNA and comparative genomic hybridization (CGH) microarray assays of human embryonic stem cells. For differentiation and cell transplantation therapy, Yang et al. report that prostaglandin E2 can promote hematopoietic differentiation from human embryonic stem cells. Li et al. discuss the features of two different neural progenitors existing in the brain and their therapeutic potential for spinal cord injuries. Sun et al. review the role of bone morphogenetic protein (BMP) signaling in regulation of neural stem cells during neural lineage development. Liu et al. review the stem cells in bone biology and their potential clinical applications for bone regeneration through collaborations among bone biologists, stem cell biologists and biomaterial scientists. Huang et al. review the contradictions of cancer stem cell theory and discuss the advancement of cancer therapy by targeting cancer stem cells. From a biotechnical perspective, Li et al. report their versatile methods for lineage tracing during embryonic stem cell differentiation by utilizing specific promoterdriven GFP expression. Yang et al. review the technical advancement of iPS cells and their potential applications in disease modeling, high-throughput drug screening and toxicological testing. Beyondmammalian stem cells, Su et al. report their study to analyze the patterns of stem cell differentiation in the processes of in vitro somatic embryogenesis in Arabidopsis; and Liu et al. review the distinct features of stem cells and their roles in shoot apical meristems.We hope these articles demonstrate some of the fascinating aspects of stem cell biology to all of our readers.

The phosphoinositide-3-kinase/Akt pathway mediates the transient increase in Nanog expression during differentiation of F9 cells

Nanog is a key determinant that maintains self-renewal and pluripotency of embryonic stem cells and represses their differentiation to endoderm. In this study, we examined the regulation of Nanog expression by phosphoinositide-3-kinase (PI3K)/Akt pathway during retinoic acid (RA)-induced differentiation of F9 embryonic carcinoma cells. Nanog protein expression was transiently upregulated up to 6 h after RA treatment and then declined. In agreement, a murine Nanog promoter reporter assay revealed that promoter activity increased during early stage of differentiation, but decreased when F9 cells became fully differentiated. RA treatment of F9 cells also led to a transient and parallel increase in both Akt and glycogen synthase kinase 3beta phosphorylations. Nanog expression was diminished in the early stage by LY294002, a PI3K inhibitor, but was not affected in the late stage despite considerable inhibition of Akt phosphorylation and endoderm marker expression by the inhibitor. These data suggest that RA-induced PI3K/Akt activation in the early stage of differentiation is required for Nanog expression, which becomes independent of PI3K/Akt signaling once the differentiation is established. Thus, Nanog expression appears to be differently regulated by the PI3K/Akt pathway depending on differentiation stage.